Whole‐mitochondrial genomes of Nannizziopsis provide insights in evolution and detection

Abstract Infectious fungal diseases can have devastating effects on wildlife health and a detailed understanding of the evolution of related emerging fungal pathogen along with the ability to detect them in the wild is considered indispensable for effective management strategies. Several fungi from the genera Nannizziopsis and Paranannizziopsis are emerging pathogens of reptiles and have been observed to cause disease in a wide range of taxa. Nannizziopsis barbatae has become a particularly important pathogen of Australian reptiles with an increasing number of herpetofauna being reported with cases of infection from across the country. Here, we present the mitochondrial genome sequences and phylogenetic analysis for seven species in this group of fungi uncovering new information on the evolutionary relationship of these emerging pathogens. From this analysis, we designed a species‐specific qPCR assay for the rapid detection of N. barbatae and demonstrate its application in a wild urban population of a dragon lizard.

with this disease (Peterson et al., 2020). Urban wildlife populations in particular have become a focal point for outbreaks and the need for effective detection and monitoring of pathogen occurrence is considered vital for mitigating the spread and to minimize any potential for transmission to humans (Ghosh et al., 2018). Molecular diagnostic tests are powerful tools for disease surveillance offering a low cost and rapid means to assist in the early detection in both captive and wild populations (Boyle et al., 2004). Such tools also enable long-term tracking of pathogens that facilitate the study of often complex host-pathogen interactions, such as how disease tolerance may effect prevalence and transmission (Seal et al., 2021;Tedersoo et al., 2019). Genomic data are valuable resources for the development of diagnostic tools enabling swift identification of target regions for designing highly specific markers, and these data can also serve as a foundation for studies on the molecular basis for pathogen evolution (DeCandia et al., 2018;Ghosh et al., 2020Ghosh et al., , 2021. Comparative genomic studies using whole-mitochondrial genomes, as opposed to metabarcoding, can help to resolve evolutionary relationships by building more robust phylogenies in newly described species and thus may enhance the effectiveness of species-specific detection methods (Tedersoo et al., 2019).
A rapid diagnostic tool to detect N. barbatae in clinical samples is currently unavailable. As Nannizziopsis fungi are typically first isolated on selective media prior to PCR and sequencing, laboratory diagnosis may involve delays of up to a week due to the slow growth of these species. The aim of this study is to gain a deeper understanding of the evolutionary relationship among this newly defined group of emerging fungal pathogens through the sequencing and analysis of their whole-mitochondrial genomes and to develop a molecular diagnostic for the specific detection of N. barbatae infections to support the study of outbreaks of this disease in wild populations.

| Sample culturing and processing
Type cultures listed in Table 1 were purchased from the UAMH Centre for Global Microfungal Biodiversity in Toronto, Canada. N. barbatae strain USC001 was isolated and sequenced previously (Powell et al., 2021 and placed on ice before transportation to the laboratory to be stored at −20°C until processed to extract DNA using the Wizard Genomic DNA Purification Kit (Promega).

| qPCR assay design
Extracted DNA from fungal cultures was checked with PCR using ITS gene fragment primers forward 5′-GCATC GAT GAA GAA CGC AGCGA-3′ and reverse 5′-GGYCA GCK CCG GCC GGGTC-3′ used in a previous study (Peterson et al., 2020) to confirm that purified DNA from the reference strains could be successfully am-

| Mitochondrial genome assembly and phylogenetics
Short-read shotgun sequencing produced a minimum 150-fold read coverage per sample that was used to reconstruct the mitochondrial genomes from six Nannizziopsis and one Paranannizziopsis reference strains. Variability in mitochondrial DNA length between species ranged from 24.5 to 30.8 Kb (Table 1)

| Species-specific qPCR performance
Using our qPCR assay, DNA from two different isolates of N. barbatae was successfully detected. Yet we could not detect any other Nannizziopsis species in this study, including the closely related N.
crocodili. These results suggest that mtDNA targets can be used to distinguish between species of Nannizziopsis fungi. A small degree of non-specific fluorescence was observed from N. dermatitidis DNA at the optimal probe annealing T m of 60°C owing to a lower amount of variation in this species at the probe target location (Figure 4).
This was resolved without sacrificing reaction efficiency by raising the annealing temperature to 63°C. The probe sequence confers the specificity in the assay as the PCR primer sequences were conserved across Nannizziopsis species and could produce an amplicon from all but N. vriesii ( Figure S1) owing to the absence of the intron in the nad1 gene in this species as mentioned above. Specificity of the assay to distinguish among the species tested was 100%.

| DISCUSS ION
In this study, we resolve the evolutionary relationship between key members of the Nannizziopsis responsible for diseases in reptiles using full-length protein sequences of 13 mitochondrial protein coding genes. We report that the high similarity between the species N. vriesii and N. dermatitidis extends across the entire mitochondrial genome with the notable exception of an intron present in the nad1 gene in N. dermatitidis. This high level of similarity suggests some taxonomic revision may be appropriate to either group these two species together or split the two strains of N. barbatae. The varying occurrence of introns was also observed between the two strains of N. barbatae in this study isolated from infected reptiles approximately 10 years apart. Each of these introns was found to contain either an LAGLI-DADG or GIY-YIG endonuclease domain motif.
These domains encode homing nucleases, suggesting these introns possess a capacity for self-splicing (Megarioti & Kouvelis, 2020). motif, it is conserved in all but one species of Nannizziopsis included in this study and could be amplified using the same primer pair in each case. We believe this DNA target is stable enough for further applications using this assay for the detection of N. barbatae.
This study furthermore offers a rapid method for the detection of N. barbatae DNA from cultured and field collected samples. Diagnostic confirmation had previously relied on cultivation and subsequent metabarcoding analysis to confirm presence of Nannizziopsis infection (Peterson et al., 2020) leading to lengthy delays in obtaining results due to the slow growth characteristics of these species (Paré & Sigler, 2016). Our qPCR assay was able to correctly distinguish DNA samples of N. barbatae from five other species of Nannizziopsis. The assay was sufficiently sensitive to detect the presence of less than one genomic equivalent per reaction, owing to the presence of multiple copies of the mitogenome for every nuclear genome. We expect there to be an average of between six to nine mitochondrial genome copies per nuclear genome for the N. barbatae isolates sequenced in this study based on differences observed in the amount of sequencing read coverage between the nuclear and mitochondrial genome. However, we caution that this may be highly variable and with only a few isolates sequenced to date, the use of higher limit of detection should be considered until sufficient data on copy number can be attained.
We also demonstrate the utility of this assay to screen a freeliving population of infected reptiles using minimally invasive swab sampling. In addition to the 67 samples that tested positive for infection with N. barbatae, there were seven instances of what appear to be false negative results out of 74 visibly diseased animals from this screening set. As these occurred only in the mildly diseased animals (rating 1 or 2,

ACK N OWLED G M ENTS
We acknowledge the Turrbal and Yugara people, as the First Nations owners of the lands where our Brisbane study site sits. We pay respect to their Elders, lores, customs, and creation spirits. In addition, we would like to thank Dr Meghan Castelli for assistance with sample processing, Dr Yordanka G. Guardiola at the UAMH Centre for Global Microfungal Biodiversity for assistance in preparing fungal samples, and Dr Pauline Wang at the University of Toronto for support in genomic sequencing.

This work was funded by an Australian Research Council Future
Fellowship (FT200100192) to CHF.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The mitochondrial genome assemblies produced in this study are available from the NCBI GenBank under the accession numbers listed in Table 1. Primer sequences used to develop the assay are listed in the Section 2 (Materials and Methods).